Method of producing lycopene through the fermentation of selected strains of blackeslea trispora, formulations and uses of the lycopene thus obtained

ABSTRACT

The method of fermentation with selected strains of  B. trispora  described in the present invention makes it possible to achieve lycopene yields higher than those currently described. The methods of isolation, purification and formulation are applicable to any natural source of lycopene, especially to submerged cultures of mucoral fungi of the genera  Blakeslea, Choanephora, Phycomyces  or  Mucor . The method of extraction makes it possible to simplify the recovery process and increase the purity of the product, relative to the methods previously described. The methods of formulation provide high added value, since they make it possible to obtain stabilized preparations of lycopene for direct application in the food and pharmaceutical fields.

FIELD OF THE INVENTION

The method of fermentation with selected strains of B. trisporadescribed in the present invention makes it possible to achieve levelsof production of lycopene higher than those currently described. Themethods of isolation, purification and formulation are applicable to anynatural source of lycopene, and especially to submerged cultures ofmucoral fungi of the genera Blakeslea, Choanephora, Phycomyces or Mucor.The method of extraction provides a simplification of the recoveryprocess and an increase in product purity relative to the methodspreviously described. The methods of formulation give high added value,as they make it possible to obtain stabilized preparations of lycopenefor direct application in the foodstuffs and pharmaceutical fields.

1. State of the Art

The carotenoids are widely distributed in nature, imparting theircharacteristic color, from yellow to dark red, to numerous naturalsubstances such as carrots, peppers, tomatoes, flowers or certainmicroorganisms, including some bacteria, fungi and photosyntheticorganisms. The carotenoids can be divided into two types: (i) purehydrocarbons called carotenes, including compounds such as β-carotene,α-carotene, γ-carotene or lycopene and (ii) molecules calledxanthophylls, which contain oxygen in various forms (hydroxyl groups,epoxy groups, etc.), including astaxanthin, zeaxanthin, capsanthin,cantaxanthin, lutein, etc. The two groups of compounds display differentbehavior with respect to their physicochemical properties and solubilityin organic solvents. All these compounds play an important role in thehuman diet, their properties having been studied extensively asantioxidants for the prevention of cancer and other human diseases andas precursors of vitamin A. It has recently been demonstrated in ratsthat lycopene inhibits the harmful effect of ferric nitriloacetate onDNA and prevents necrosis of the liver [Matos H.R. et al. (2001) Arch.Biochem. Biophys. Vol. 396]. In addition, owing to their colorationsfrom yellow to red, the carotenoids are of considerable commercialimportance as colorants and food additives on account of theirbeneficial effects on health and their attractive colors [Ninet L. andRenaut J. (1979) In: Peppler H J., Perlman D. (eds). MicrobialTechnology, 2nd Edition, Vol. 1, Academic Press, New York, pp. 529-544].

Lycopene (C₄₀H₅₆) is an intermediate in the biosynthetic pathway ofβ-carotene and the xanthophylls. It has a molecular weight of 536.85 andthe following molecular formula:

Lycopene

As well as acting as an antioxidant, lycopene prevents cardiovasculardiseases and some types of cancer and is active in growth control[Giovannucci et al. (1995) J. Nat. Cancer Inst. 87: 1767-1776; Stahl W.and Sies, H. (1996) Arch. Biochem. Biophys. 336: 1-9; Clinton, S K.(1998) Nutr. Rev. 56: 35-51]. This has led to increased demand on thepart of consumers. Production of lycopene as a high-purity compound hasbeen linked in the past to chemical synthesis [U.S. Pat. No. 5,208,381;U.S. Pat. No. 5,166,445; U.S. Pat. No. 4,105,855; U.S. Pat. No.2,842,599]. However, alternative routes now exist, based on sources oflycopene of natural origin and special extraction processes.

The production of carotenoids by microbial biosynthesis is a classicexample of competition between chemical and biological processes.Lycopene preparations of biological origin are obtained from tomato [PCTWO 97/48287, EP 608027] or by fermentation of mucoral fungi of thegenera Phycomyces, Blakeslea and Choanephora [GB 1008469, U.S. Pat. No.3,097,146, U.S. Pat. No. 3,369,974, JP 73016189, JP 73016190, RU2102416, WO 00/77234]. To achieve a maximum yield of carotenoids with B.trispora it is necessary to ferment the (+) and (−) strains together[Ciegler, A. (1965) Advances in Applied Microbiology 7: 1-34; Plempel,M. (1965) Planta 65:225-231; Sutter, R P. and Rafelson, M E. (1968) J.Bacteriology 95: 426-432]. The increase in yield of carotenoids in mixedcultures is related to the production of a family of acid compoundscalled factor β or trisporic acids [WO 00/77234, Caglioti L. et al.(1966) Tetrahedron Supplement 7: 175-187]. For the biosynthesis oftrisporic acids, the β-carotene produced by the (+) and (−) strains ismetabolized by both to retinal and subsequently to 4-dihydrotrisporol.The (+) strain utilizes the 4-dihydrotrisporol as substrate for formingdihydrotrisporic acid and its methyl ester (methyl-4-dihydrotrisporate).For its part, the (−) strain metabolizes the 4-dihydrotrisporol totrisporol. Finally, the methyl-4-dihydrotrisporate is converted totrisporic acid by the (−) strain and the trisporol is converted totrisporic acid by the (+) strain. This description of the biosynthesisof the trisporic acids is a simplification, since during the processmany co-metabolites are generated, some of which are common to bothstrains (+) and (−), but others are specific to one of them. Therelative quantities of these co-metabolites vary depending on thestrains.

The biosynthetic pathway of β-carotene (see scheme 1) has been describedin fungi that are related phylogenetically to B. trispora such asPhycomyces blakesleeanus and Mucor circinelloides [Arrach N. et al.(2001) Proceedings of the National Academy of Sciences USA 98:1687-1692; Velayos A. et al. (2000) European Journal of Biochemistry267: 5509-5519]. At least three enzymes are necessary for saidbiosynthesis: (i) phytoene synthase, which joins together two moleculesof geranylgeranyl pyrophosphate to form phytoene, (ii) phytoenedehydrogenase, which introduces four double bonds into the phytoenemolecule to synthesize lycopene, and (iii) lycopene cyclase, which,using lycopene as substrate, forms the rings located at the two ends ofthe β-carotene molecule. It was concluded on the basis of analysis ofmutants of B. trispora that the biosynthetic pathway of β-carotene inthis fungus is similar to that described for P. blakesleeanus [Metha B.J. and Cerdá-Olmedo E. (1995) Applied Microbiology and Biotechnology 42:836-838]. In the case of P. blakesleeanus, the yellow color of itsmycelium can be altered by mutation, giving rise to strains withmycelium colored red, white or various shades of yellow. The red mutantsaccumulate lycopene, whereas the white ones lack production ofcarotenoids or accumulate phytoene. For production of lycopene it isnecessary to have strains of B. trispora that lack lycopene cyclaseactivity, or alternatively chemicals that inhibit said enzymaticactivity must be added to the fermentation medium.

Patents GB 1008469, U.S. Pat. No. 3,097,146, U.S. Pat. No. 3,369,974, JP73016189, JP 73016190, RU 2102416 and WO 00/77234 describe theproduction of lycopene by means of fermentation of mucoral fungi such asPhycomyces, Blakeslea and Choanephora . Patents GB 1008469 and U.S. Pat.No. 3,097,146 describe methods of fermentation of B. trispora based oncontrol of the pH between values of 7.0 and 9.5, obtaining yields of99.7 mg/l of lycopene after 7 days of fermentation. Patents JP 73016189and JP 73016190 describe methods of production of lycopene with mucoralfungi based on the addition of tertiary amines. Patent RU 2102416describes the addition of aminomethylpyridines and tobacco residues forinducing the accumulation of lycopene. As well as the substancesdescribed in said patents, the use of other nitrogenated heterocyclicbases for blocking the synthesis of carotenoids at the lycopene levelhas been published: nicotine [JP 09313167], imidazole, pyridine,morpholine, quinoline and some substituted derivatives [U.S. Pat. No.3,369,974; Ninet L., Renaut J. (1979) In: Peppler H J, Perlman D (eds).Microbial Technology, 2nd Edition, Vol. 1, Academic Press, New York, pp.529-544]. Moreover, mutants of B. trispora that accumulate lycopenewithout the need to add tertiary amines have been described [Mehta B. J.and Cerdá-Olmedo E. (1995) Appl. Microbiol. Biotechnol. 42: 836-838].

In addition to the aforementioned mucoral fungi, production of lycopenehas been described with algae [JP 09313167 and JP 2000152778], byfermentation of Streptomyces chrestomyceticus var. rubescens [U.S. Pat.No. 3,467,579] and by modifying the biosynthetic pathway of carotenoidsof Flavobacterium sp. R1534 [U.S. Pat. No. 6,124,113].

Lycopene can be obtained from plant products such as: tomato, carrot,peppers, vegetable oils, etc. Thus, patent WO 97/48287 describes amethod for the preparation of lycopene-rich oleoresins from tomatoes bypressing the tomatoes until the pulp is obtained, extraction of lycopenefrom the pulp with organic solvents and subsequent elimination of thesolvent by evaporation, giving rise to an oleoresin with a lycopenecontent in the range 2-10%. Similar methods of obtaining oleoresins richin carotenoids in general and lycopene in particular from plants andoils are described in various patents, such as in U.S. Pat. No.5,245,095 and EP 580745, by precipitation with calcium salts, in U.S.Pat. No. 5,019,668, using a method of transesterification with oilsfollowed by distillation, in WO 95/16363, which describes thefractionation of the tomato into various fractions that include anoleoresin rich in carotenoids, and in PCT WO 90/08584, which describesthe extraction of lycopene by using fluids in a supercritical state,although the extract obtained is a mixture of various carotenoids andthe extraction yields are very low owing to their low solubility.

In all these cases, owing to the low concentration of lycopene in thesenatural products and the intracellular location of this compound incertain organelles such as chloroplasts or chromoplasts, the extractionyields and the purity of the product obtained are low, obtainingoleoresins rich in lycopene or dehydrated raw products together withvarying amounts of other carotenoid or non-carotenoid compounds. In themajority of cases the methods of extraction described requirepreparation of the fruit by milling or pressing to facilitate extractionof the solvent and thus release the lycopene-rich intracellularcontents. Finally, most of the processes described in these patentsrequire the use of organic solvents that are present as traces in theoleoresin obtained. Furthermore, patent IL 107999 describes thepreparation of oleoresins that are very rich in lycopene from tomatopulp, although, as previously, the product obtained does not consist oflycopene crystals of high purity, but of lycopene-rich lipidconcentrates.

On the other hand, patent WO 97/15554 describes the extraction ofcarotenoids of plant origin from carrots and tomatoes, which includelycopene, by isolation of chloroplasts and chromoplasts, followed bydigestion of said organelles with hydrolytic enzymes of proteins such aspectins and/or proteases that make it possible to release the lycopenebound to various structural proteins. By subsequent alkaline treatmentand extraction with alcoholic mixtures of low molecular weight it ispossible to obtain lycopene extracts with a richness and purity greaterthan the oleoresins, though without obtaining purified crystals oflycopene but lycopene-rich raw extracts. Similarly, concentratedextracts of lycopene are obtained in patent EP 608027 A2 by isolation oftomato chromoplasts in which lycopene occurs in crystalline form. Theseextracts from lycopene-rich tomato chromoplasts are used directly ascolorants without subsequent extraction of the lycopene crystals,avoiding the color change of the lycopene during extraction and makingthe use of organic solvents unnecessary. In accordance with the methoddescribed in this patent, it is not possible to obtain pure lycopene incrystalline form suitable for use in foodstuff or pharmaceuticalcompositions, but only as food colorant in dehydrated, freeze-dried orfrozen form.

Certain carotenoid-rich micro-algae of the Dunaliella type are anotherimportant source of lycopene. There are various methods of extractingcarotenoids, and lycopene in particular, from these organisms, as isreflected in patents U.S. Pat. No. 5,378,369, U.S. Pat. No. 4,713,398and U.S. Pat. No. 4,680,314, by extraction with organic solvents(chlorocarbons, hydrocarbons, etc.) or edible oils (DE 4342798). Adifferent process is described in PCT WO 98/08584, where a lycopeneextract is obtained using CO₂ in a supercritical state, although theextract thus obtained is of low purity with respect to lycopene.

Lycopene can also be obtained from certain mucoral fungi such asBlakeslea, Choanephora, Phycomyces or Mucor by fermentation in a liquidmedium, offering as an advantage over the production of lycopene fromplant products or algae the increased concentration of this compound, insome cases above 5 wt. % relative to the quantity of dry biomass,concentrations that are higher than those obtained from the best plantvarieties, as well as the possibility of the biotechnologicaldevelopment of overproducing strains of these microorganisms, either bytechniques of classical mutagenesis or by the application of newtechnologies of molecular biology that permit the genetic manipulationof these microorganisms, increasing the concentration and yield oflycopene, and eliminating the production of other structurally relatedcarotenoids.

As already mentioned, the preparation of crystalline lycopene of highpurity from natural sources generally requires a stage of extractionwith organic solvents or fluids in a supercritical state and thenvarious additional purification stages such as chromatography, processesof adsorption and elution and stages of precipitation orcrystallization, as described for example in patents U.S. Pat. No.3,369,974, EP 818255 and EP 242148. In the majority of cases in whichthese stages of subsequent purification are not used and crystallizationis carried out directly from the extract by evaporation of the solventuntil the solubility is overcome, the purity of the product obtained isvery low and it is subsequently necessary to carry out processes ofrecrystallization of the lycopene obtained, with the added difficultythat the low solubility of the product means a large quantity of solventmust be used to achieve the stage of recrystallization, which leads inaddition to a low yield (NL 6411184, U.S. Pat. No. 4,439,629).

Patent application WO 96/13178 describes the preparation of concentratesof stabilized crystalline lycopene in a foodstuffs-compatible liquidmedium in which lycopene is insoluble, such as ethylene glycol, ethanolor glycerol, obtaining, by milling, small crystals (1-3 microns) oflycopene suspended in a liquid medium. Moreover, patent application WO98/43620 describes a method of isolating lycopene crystals from anoleoresin by saponification of various triglycerides and phosphonates athigh temperature and then dilution with water, obtaining lycopenecrystals of purity between 75% and 95%. A similar method was recentlydescribed for the preparation of crystals of β-carotene of high purityin PCT WO 98/03480 by washing with water, alcohols of low molecularweight or acetone, though its application for obtaining lycopenecrystals of high purity has not been described.

The instability of the carotenoids in crystalline form is well known,and one method of stabilizing them is the preparation of oilydispersions. Moreover, it is believed that carotenoids dispersed in oilare absorbed more easily by the body. An alternative method for thestabilization of unstable compounds is their microencapsulation instarch matrices. Thus, patents U.S. Pat. No. 2,876,160, U.S. Pat. No.2,827,452, U.S. Pat. No. 4,276,312 and U.S. Pat. No. 5,976,575 describea considerable increase in the stability of various compounds, includingthe carotenoids, by encapsulating them in a starch matrix.

One of the main difficulties in using carotenoids in the field ofcolorants is their zero solubility in water, since many of theirapplications take place in aqueous media. This problem of solubility wasmentioned in document U.S. Pat. No. 3,998,753, and was solved bypreparing solutions of carotenoids in volatile organic solvents, such ashalogenated hydrocarbons, and emulsifying them with an aqueous solutionof sodium lauryl sulfate.

U.S. Pat. No. 5,364,563 describes a method of producing a preparation ofcarotenoids in powder form, which involves forming a suspension of acarotenoid in oil with a high boiling point. The suspension issuperheated with steam for a maximum period of 30 seconds in order toform a solution of carotenoid in oil. Next, this solution is emulsifiedover an aqueous solution of a colloid and then the emulsion isspray-dried.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes a series of methods for obtaining highyields of lycopene with the fungus B. trispora , as well as methods forits recovery and formulation. The invention consists of (i) the designof methods for obtaining and selecting mutants of B. trispora that areoverproducers of lycopene, (ii) the development of improved conditionsof fermentation, (iii) the establishment of processes for recoveringlycopene from the mycelium and (iv) the achievement of formulations thatovercome the problems of stability and solubility in various media,present in the state of the art. B. trispora is a fungus that is ofgreat industrial importance for the biotechnological production oflycopene. In fact, said process proves to be competitive with thesynthetic process used industrially at present.

With the aim of obtaining strains that are overproducers of lycopene, inthe first place a mutagenic method was developed for the (+) and (−)strains of B. trispora with the mutagenic agents ethylmethane sulfonate(EMS) and N-methyl-N′-nitro-N-nitrosoguanidine (NTG). The suspensions ofspores for mutation were obtained from slants with YpSs medium. Thespores were resuspended by adding 10 ml of a solution of Triton X-100 at0.1% to each slant. The mycelium residues were removed by filtrationthrough a nylon filter with a pore size of 20 μm. The concentration ofspores in the suspension was adjusted to 10⁶ spores/ml. The method ofmutation with EMS consisted of incubating 10⁶ spores/ml in a 3% EMSsolution in 0.1 M sodium phosphate buffer pH 7.0 at room temperature for60 minutes, achieving mortality rates of around 99%. The mutated sporeswere washed three times with 0.1% Triton X-100 and centrifuged at 3000rpm at 15° C. for 2 minutes. The method of mutation with NTG consistedof incubating 106 spores/ml in a solution that contained 250 μg/ml ofNTG and 0.1 M sodium citrate buffer pH 5.0 at room temperature for 30minutes, achieving mortality rates of around 95%. The mutated sporeswere washed three times with 0.1% Triton X-100 and centrifuged at 3000rpm at 15° C. for 2 minutes. Petri dishes containing Sutter IV solidmedium supplemented with 0.1% Triton X-100 were seeded with the mutatedspores and incubated at 25° C. for 4 days to obtain isolated colonies.

The strategies employed for selecting lycopene-overproducing (−) strainsof B. trispora were as follows: (i) the use of trisporic acids and (ii)the color intensity of the colony. Selection of lycopene-producingmutants by addition of trisporic acids consisted of placing filtersimpregnated with trisporic acids over the colonies obtained from mutatedspores. The trisporic acids were obtained from a mixed culture of the(+) and (−) strains of B. trispora . The colonies plus filters wereincubated at 25° C., and it was observed that the lycopene-producingmutants acquired a deep red color, in contrast to the producers ofβ-carotene which were colored orange. Applying this method with the CMA3(−) strain, the LMA1 (−) strain was selected (Scheme 2). Selection oflycopene-producing mutants as a function of the color intensity of thecolony was carried out in the following way: The CMA1 (−) strain(producer of β-carotene; see Scheme 2) was mutated and the mutatedspores were grown on plates of YEPDA solid medium. Next, those coloniesthat possessed a deeper yellow-orange color than the CMA1 (−) parentstrain were selected. In this way 2 colonies with a deep yellow-orangecolor were isolated (designated CMB1 (−) and CMB2 (−))

Phylogeny of the (−) strains of B. trispora obtained from B. trisporaVKPM F-208 (−) using methods of mutation and selection. UV: ultraviolet;SN: natural selection; NTG: N-methyl-N′-nitro-N-nitrosoguanidine; EMS:ethylmethane sulfonate.

Selection of lycopene-overproducing mutants of B. trispora (+) waseffected by growing mutated spores in Petri dishes containing Sutter IVsolid medium supplemented with 0.1% imidazole. Next, a portion of eachof the colonies was transferred to a dish of PDA in which B. trispora(−) had previously been seeded. The level of lycopene production insolid medium was determined as a function of the intensity of colorationin the zone of intersection of the colony of the (+) strain with that ofthe (−) strain. In this way the B. trispora CPA1 (+) strain was selected(Scheme 3), which gave rise to a higher yield of lycopene in mixed solidcultures with a series of (−) strains. The level of production of the B.trispora CPA1 (+) strain was then analyzed in mixed culture in liquidmedium.

The system of symbols employed for designating the selected strains isas follows:

CM: Carotene minus (−).

LM: Lycopene minus (−).

CP: Carotene plus (+).

LP: Lycopene plus (+).

The relationship between parent generations follows alphabetical order:A is the parent of B, B is the parent of C, and so on. The number afterthe letters corresponds to the number of the mutant. For example, thedesignation CMA1 (−) signifies that it is a carotene-producing strain(C), minus (M), parental of CMB and mutant number 1. Similarly, CMA1(−), CMA2 (−), CMA3 (−) and CMA4 (−) correspond to mutants 1, 2, 3 and 4of the same generation.

Phylogeny of the B. trispora (+) strains obtained from B. trispora VKPMF-117 (+) using methods of mutation and selection. UV: ultraviolet; SN:natural selection; NTG: N-methyl-N′-nitro-N-nitrosoguanidine; EMS:ethylmethane sulfonate.

The (+) and (−) strains of B. trispora selected in solid medium werefermented in a flask with the aim of determining the level of productionof lycopene in liquid medium and mixed culture. For this, separateflasks of inoculum were seeded with the strains B. trispora CPA1 (+) andB. trispora CMB2 (−) and then mixed fermentation of both strains waseffected in a flask. At the start of fermentation (0-50 hours) aninhibitor of the enzyme lycopene cyclase was added with the aim ofblocking the biosynthetic pathway at the lycopene level (for exampleimidazole at a concentration of 0.7-0.8 g/l). At the end of fermentation(about 6 days), the mycelium of B. trispora was lyzed by vortexagitation, the lycopene was extracted with organic solvents (e.g.acetone) and its concentration and purity were determined by HPLC. Theyields obtained were 3.0 g/l. The same type of fermentation was carriedout with the strains B. trispora CPA1 (+) and B. trispora LMA1 (−),except that in this case it was not necessary to add an inhibitor of theenzyme lycopene cyclase. The yields obtained with these strains in mixedculture were 1.2 g/l.

The CPA1 (+) and CMB2 (−) strains were cultivated in a semi-commercialfermenter with the aim of determining the lycopene yield. For this, theywere grown separately in flasks, were transferred separately tointermediate growing tanks and finally they were fermented together.Between 25 and 35 hours of fermentation, imidazole was added asinhibitor of the enzyme lycopene cyclase. The fermentation was incubatedfor 100-140 hours. The average value of lycopene yield obtained in aseries of different fermentations of the CPA1 (+) and CMB2 (−) strainswas 3.4 g/l.

The CPA1 (+) and LMA1 (−) strains were cultivated in a semi-commercialfermenter with the aim of determining the lycopene yield withoutaddition of inhibitors of the enzyme lycopene cyclase. Fermentation wascarried out as indicated previously for the CPA1 (+) and CMB2 (−)strains, but without adding imidazole. The average value of lycopeneyield obtained in a series of different fermentations of the CPA1 (+)and LMA1 (−) strains was 1.6 g/l.

A higher yield in this fermentation stage is obtained by controlling theage of the vegetative stages of growth of the strains of B. trispora .Thus, the cultures used as inoculum have an age of 30-60 hours,preferably of 48 hours, both for the (+) and the (−) strains, butvarying the number of spores seeded: 800-1000 spores/ml and 40 000-60000 spores/ml, respectively. Incubation is carried out at about 25° C.with 0.1% v/v of each inoculum seeded in the primary culture phase. Theage of said primary cultures varies in the range 30-60 hours, preferably36-48 hours, at temperatures in the range 26-28° C. Then the (+)/(−)primary phases are mixed in the ratio 1/10 v/v and the fermenters areseeded 10-20% v/v with the mixture of said phases.

In view of the intracellular characteristics of the carotenoid componentbiosynthesized in the fermentation, the method of recovery from theculture medium, prepared as claimed in the usual methods, involves as afirst stage the separation of the biomass from the culture medium. Thisseparation can be effected by the established methods of filtration,employing the usual technologies with filters, whether belt filters,rotary filters, press filters etc., in which a barrier consisting of thefilter cloth separates the biomass and allows the liquid phase withoutbiomass to pass, or centrifugation, in which, by utilizing the densitydifference between the culture medium and the biomass (normally ofhigher density), a machine such as a centrifugal separator, decanter orthe like is employed, in which the heavy phase becomes concentrated andseparates from the liquid phase with the least possible quantity ofbiomass. One of the objectives of this stage is to reduce losses andoptimize the characteristics of each phase, achieving the greatestquantity of biomass with the highest content of dry residue andeliminating most of the fermentation medium, with the smallest quantityof active material.

The resulting wet mycelium contains more than 95% of the carotenoidsproduced in fermentation, preferably more than 97% and more preferablymore than 99%. The content of carotenoids in the aqueous phase istherefore less than 5%, preferably less than 3% and more preferably lessthan 1%. With this wet mycelium it would be possible, by means of thesubsequent stages, to separate the lycopene. However, it has been foundthat, in connection with fermentation, this mycelium still has arelatively high percentage of lipophilic components, between 15 and 20%(fatty acids and oils), which cause problems of purification in laterstages, so it becomes necessary to introduce a stage of purification ofthe biomass at this point. The purification stage involves resuspendingthe biomass in alcohol: methanol, ethanol, propanol, isopropanol, or anyother alcohol in which the solubility of lycopene is very low, to asufficient extent to achieve maximum purification of the lipidcomponents. Thus, the wet mycelium is resuspended with a quantity ofalcohol ranging from 1 ml/g to 10 ml/g of wet mycelium. The temperatureof resuspension varies between 0° C. and the boiling point of thealcohol, preferably between 10 and 50° C. The contact time is in therange from 5 minutes to 24 hours. The alcoholic resuspension thusprepared is filtered or centrifuged, so that the solids content in thefiltrate or clarified liquid is practically zero. The resulting wetmycelium, which will contain alcohol plus water in varying proportions,contains more than 93% of the carotenoids produced in fermentation,preferably more than 95% and more preferably more than 97%.

In the supernatant or filtrate, which contains residues of culturemedium and alcohol, the carotenoids content is less than 2%, preferablyless than 1%, relative to the initial culture medium. This treatmentwith alcohol makes it possible to remove a number of alcohol-solublelipophilic substances, in varying amounts depending on thecharacteristics of the culture medium used, effecting a pre-purificationwhich will make it possible to obtain a crystalline final product ofhigh purity. Furthermore, by removing a varying proportion of water fromthe initial wet mycelium, the subsequent drying process is greatlyfacilitated. By mixing the culture medium directly with the alcohol andmaintaining a minimum contact time, a purification effect is achievedequivalent to that described previously, so that the process issimplified by the elimination of one operation of solid-liquidseparation. The culture medium/alcohol ratio can vary from 1/0.5 to 1/5,and is preferably between 1/1 and 1/3. The temperature of the mixturevaries between room temperature and the boiling point of the mixture,and preferably between room temperature and 60° C.

The dewatered/purified mycelium is dried. Drying can be carried out bythe usual batch or continuous methods. The drying temperature variesbetween room temperature and 150° C., preferably it should not exceed60° C. and more preferably it should be below 50° C. The drying timedepends on the temperature used, and varies between 1 hour and 72 hours.Owing to possible decomposition of these carotenoids by oxidation byatmospheric oxygen, it is best to effect this drying operation in theabsence of oxygen, either under a nitrogen atmosphere or at least undervacuum. The fact that the carotenoid product is intracellular means thatconditioning of the purified biomass is required, either by drying plusmilling, drying plus disintegration or disintegration of the biomass,which promotes mixing with solvents and facilitates solvent extraction.So that the solvent has good access to the carotenoid to be extracted, aprior operation of breaking of the mycelium is necessary, to maximizethe area of contact. The optimum particle size of the dry, brokenmycelium must be less than 3 mm, preferably less than 1 mm and morepreferably less than 0.5 mm.

Milling can be carried out on the dry product, by means of mechanicalsystems with swiveling or fixed parts: hammers, screens, etc., bypassage through rotating cylinders pressing on one another (compactionor extrusion). It is also possible to effect drying and milling in asingle stage by means of a flash (instantaneous) drying system in a jetmill, where the wet product is fed to a recirculating gas stream at hightemperature, in such a way that the residence time is the minimum tovaporize the content of liquid components, and the product istransported, as the densities vary, to a cyclone where it is recovered.During the drying time and in the drying path, there is also an effectof homogenization as the particles impinge on the walls.

Various organic solvents can be used for extracting the lycopene from amycelium conditioned in the manner described. This invention will referto the use of solvents of foodstuff grade that are regarded as natural,such as acyl esters, preferably ethyl, propyl, isopropyl, butyl orisobutyl acetates, which combine reasonably high solubility for thecarotenoid components with their compatibility as solvents included inthe Group of Class III of the ICH. These solvents are permissible bothat national and at community level, in the pharmaceutical and in thefoodstuffs field (RDL12/04/90 and RDL16/10/96). As claimed in the ICH,the residual solvents content must be below 5000 ppm, preferably below1000 ppm and more preferably below 100 ppm, based in each case on thedry matter of the liquid mixture. The extraction temperature variesbetween room temperature and the boiling point of the solvent,preferably between 50° C. and 80° C. The extraction time will be theminimum necessary to achieve dissolution, between 1 second and 1 hour,preferably between 1 minute and 15 minutes. The quantity of solvent useddepends on the temperature and on the carotenoids content of themycelium, varying between 5 ml/g and 30 ml/g of biomass. The number ofextractions varies from 1 to 3. The quantity of carotenoids extracted isgreater than 85%, preferably greater than 90% and more preferablygreater than 95%.

Once obtained, the carotenoid-rich extract must be concentrated to adefined volume. The final concentration of carotenoids in the solventafter concentrating is preferably between 10 and 50 g/l. The temperatureof concentration must be below 80° C., preferably below 70° C. and morepreferably below 50° C. Once the extract has been concentrated to therequired volume it is necessary to add an insolubilizer of thecarotenoids, specifically an alcohol and more specifically methanol,ethanol, propanol, isopropanol or any other alcohol in which thesolubility of the lycopene is very low, so that the yield of crystallinelycopene increases considerably. Addition of the alcohol also has apurifying effect. The crystallization time varies between 15 min and 24hours, preferably between 1 h and 12 h and more preferably between 3 and8 hours. The crystallization temperature must be below 25° C.,preferably below 5° C.

Separation of the crystals from the crystallization liquor can beeffected by filtration or centrifugation, displacing the crystallizationliquor in which the crystals are immersed by washing with the samealcohol as employed for insolubilization. The crystals obtained aredried under vacuum at room temperature for at least 1 h until a residualsolvents content is obtained that meets the specifications laid down bythe legislation mentioned earlier and which, in the case of lycopene,stipulates a loss on drying of less than 0.5%.

The purity of the crystals obtained corresponds to a titer above 95%,determined by spectrophotometry by reading the absorption at 472 nm of asolution of the crystals in n-hexane (E1% 1cm=3450), with a content ofother carotenoids below 3%. The content of cis lycopene is below 3%.

The method of this invention is especially suitable for the recovery ofcrystalline lycopene from a microbial source, preferably algae, fungi oryeasts, more preferably from fungi of the Mucorales order, and morepreferably B. trispora. The exceptional purity achieved for the crystalsobtained by the present methodology and the use of solvents that areregarded as natural means that these crystals can be used. in the food,pharmaceutical or cosmetics industry.

The crystalline product obtained by the methodology described in thisinvention can be packed in opaque containers which preventphotodegradation of the product, in the absence of oxygen (inertatmosphere or vacuum) to prevent oxidation and at temperatures between 0and 5° C. The product, properly packed, can be handled and marketed “asis”. However, it is advisable to increase its stability by subsequentstages of formulation or finishing, involving the addition ofantioxidants that make it possible to obtain a finished product with ashelf life greater than 6 months when properly packed.

Another essential object of this invention is a method of preparation oflycopene that includes its formulation in various presentations as afunction of the characteristics of the application for which thelycopene is to be used. A first application, called microcrystallinesuspension of lycopene in vegetable oil, consists of premixing of theaforesaid crystalline lycopene with a variable amount of vegetable oil.The type of vegetable oil can be very varied, the commonest though notthe only ones being sunflower oil, olive oil, corn oil, soya oil,cottonseed oil, etc. The dosage of lycopene will be a function of thefinal strength required, the commonest values being suspensions with alycopene content between 5 and 60%, preferably between 10 and 30%. Toincrease the stability of the mixture, the usual liposolubleantioxidants are added, such as natural tocopherols, and preferablyD,L-alpha-tocopherol. The proportion of this compound varies between 0.2and 15% relative to the weight of lycopene, preferably between 0.5 and5%. For the formulations that contain lycopene to have a satisfactoryphysiological activity, it is necessary to reduce the size of thelycopene crystals. This is achieved with the usual milling systems thatare suitable for liquid mixtures. A special object of this invention areball mills that permit reduction of the size of the crystals below 10microns, preferably below 5 microns, and even more preferably below 2microns, using microspheres with a diameter between 0.5 and 0.75 mm.Nevertheless, the crystal size can vary as claimed in the particularapplication of the suspension, employing appropriate spheres and millingconditions in each case. This crystal size will also determine therheological properties of the mixture, especially its viscosity, whichcan also be adjusted as claimed in requirements. These microcrystallinesuspensions of lycopene in oil are suitable for applications of lycopenein lipophilic environments: margarine, butter, creams, etc.

A second application, called cold-water-dispersible (CWD) lycopeneformulation, is based on dissolution of the lycopene in an organicsolvent and its subsequent microencapsulation in modified starches. Thesolvents that are most suitable for effecting this dissolution, as thismolecule exhibits high solubility, are chloroform, benzene, toluene,etc. Methylene chloride is especially suitable. However, owing to thehalogenated character of the latter it is possible to use food-gradesolvents that are regarded as natural, such as acyl esters, preferablyethyl, propyl, isopropyl, butyl, isobutyl and other acetates, whichcombine reasonably high solubility for the carotenoid components withtheir compatibility as solvents included in the Group of Class III ofthe ICH. The concentration of lycopene in the organic solvent can varybetween 1 and 50 g/l, preferably between 10 and 30 g/l. The dissolutiontemperature can vary between room temperature and the boiling point ofthe solvent, preferably between 20 and 130° C. The fact that thepercentage of cis lycopene is a function of the temperature/time ratioin the operation of dissolution of the lycopene in the organic solventmeans that if we wish to obtain a product with a low content of thisisomer, either a low dissolution temperature is used, or otherwise avery short dissolution time. Thus, to achieve low levels of cis, if thesolvent employed is methylene chloride, dissolution can be carried outat 20-35° C. for a time of between 1 and 15 minutes. If, on the otherhand, the solvent is isobutyl acetate, dissolution will preferably beeffected between 70 and 130° C. for a few seconds. However, if thelevels of cis isomer are not relevant, dissolution can be carried outwithout restriction on its conditions other than attainment of totalsolubility at the molecular level of the lycopene in the solventemployed. To increase the stability of the final formulation, one or amixture of several antioxidants are dissolved together with the lycopenein the organic solvent; these antioxidants are preferably those such astocopherol, ascorbyl palmitate, etc., each of them in a proportionbetween 1 and 30%, preferably between 10 and 20%, relative to the weightof lycopene. It is also possible to incorporate vegetable oil in themixture: sunflower oil, olive oil, corn oil, soya oil, cottonseed oil,etc., for the purpose of promoting dissolution of the lycopene, andimparting additional stability to the preparation. The lycopene/oilratio can vary between 10/1 and 1/10.

The solution of lycopene thus obtained is mixed and emulsified with anaqueous solution that contains an emulsifying agent, for examplemodified starch, more concretely esters derived from starch, preferablyoctenyl succinates derived from starch of various molecular weights, andespecially, but not exclusively, Purity Gum 2000 ® from National Starchor Cleargum CO 01® from Roquette, and a microencapsulating agent,consisting for example of modified starch, more concretely estersderived from starch, preferably octenyl succinates derived from starchof various molecular weights, and especially, though not exclusively,HiCap 100® or Capsul® from National Starch. The proportions in which theemulsifying agent and the microencapsulating agent are mixed can varybetween 5/95 and 95/5, preferably between 25/75 and 75/25, morepreferably between 40/60 and 60/40. The water content of each of thecomponents of the mixture of emulsifying agent and microencapsulatingagent is variable, and can be between 1 and 30%, preferably between 5and 20%, and more preferably 10%. The mixture of aqueous and organicphases is emulsified and the emulsion obtained is homogenized employingpressure-differential homogenization systems of the Mantón Gaulin orMicrofluidizer type, as commonly used, and preferably by homogenizationby tangential friction, for example with an emulsifier of theUltraturrax type for a time that varies as a function of the energysupplied by the equipment and the volume of mixture to be emulsified,with the aim of obtaining an average micelle size smaller than 10microns, preferably smaller than 2 microns and more preferably between0.1 and 1 micron.

Once the emulsion has formed, evaporation of the organic solvent iseffected, preferably by vacuum distillation at a temperature below 50°C. As evaporation of the solvent proceeds, microcrystallization of thelycopene takes place in the starch matrix. Once the solvent hasevaporated, evaporation continues, with successive additions of wateruntil a residual solvents content is obtained that complies with thespecifications on maximum concentration laid down by the legislation anda dry residue that is suitable for the type of drying that is to beapplied to this liquid mixture. Suitable values of dry matter in thesuspension of microencapsulated lycopene are between 1 and 30%, andpreferably between 10 and 20%.

In accordance with the present invention, it is found that both themethod of drying by high-temperature pulverization (atomization) and themethod of fluidized-bed pulverization (granulation) are suitable fordrying the aqueous suspension of lycopene obtained. Another alternativewould be freeze-drying. As claimed in the method of drying byatomization, suitable inlet temperatures of the drying air would bebetween 100 and 200° C. whereas the outlet temperatures would be between60 and 120° C. The atomized product has a particle size between 10 and100 microns. With the aim of increasing the particle size and reducingthe area available, and thus increasing the product's oxidationresistance, the atomized product can be agglomerated by pulverization ofa solution of one of the modified starches used in the formulation, orthe suspension of microencapsulated lycopene itself, within a fluidizedbed of said atomized product, which makes it possible to attain particlesizes that vary between 50 and 500 microns, preferably between 200 and300 microns.

The method of granulation involves the use of a fluidized-bed granulatorin which seed material is placed, which can be a typical inert material,such as particles of sugar, or fine powder of the actual material to bedried, obtained in previous granulation processes or in a spray-dryingprocess. The particles are kept in motion by means of air, and thetemperature of the bed is maintained between 30 and 90° C., preferablybetween 50 and 80° C. The suspension of microencapsulated lycopene issprayed by means of air preheated to a temperature between 20 and 140°C. within the fluidized bed, at a rate that ensures that the particlesthat will be coated do not become too wet and do not form lumps. Thegranulated product has a particle size between 100 and 2000 microns,preferably between 100 and 800 microns and more preferably between 100and 300 microns. Once pulverization by one or other method has beencompleted, the particles can be coated. This coating can be effectedwith approximately 0.5-10% in dry weight of aqueous solutions of varioussugars or even of one or a mixture of the starches that make up theformula that is the object of the present invention.

Deposit of Microorganisms in Accordance with the Treaty of Budapest

The strains of Blakeslea trispora have been deposited, in accordancewith the provisions of the Treaty of Budapest, in the Russian NationalCollection of Industrial Microorganisms (VKPM), GNII Genetika, DorozhnyProezd 1, Moscow 113545 (Russia), with the following numbers and dates:VKPM F-117 on 12.21.1979, VKPM F-208 on 12.20.1979, VKPM F-551 on11.19.1992, VKPM F-674 on 11.19.1992, VKPM F-726 on 01.21.1997, VKPMF-727 on 01.21.1997, VKPM F-736 on 10.07.1997, VKPM F-741 on 01.28.1998,VKPM F-744 on 01.28.1998 and VKPM F-816 on 12.13.2000.

The following examples describe the present invention in detail andwithout limitation.

EXAMPLE 1

Strategies for Mutation of the (+) and (−) Strains of B. trispora

Firstly a mutagenic method was developed for the (+) and (−) strains ofB. trispora , for which the following were analyzed: (i) various typesof mutagenic agents, (ii) concentration of the mutagen, (iii)concentration of spores, (iv) incubation pH, and (v) treatment time. Inthis way, ethylmethane sulfonate (EMS) andN-methyl-N′-nitro-N-nitrosoguanidine (NTG) were selected as mutagenicagents.

The suspensions of spores to be mutated were obtained from slants withYpSs medium, which. has the following composition: yeast extract 4 g/l,soluble starch 15 g/l, K₂HPO₄ 1 g/l, MgSO₄.7H₂O0.5 g/l and agar 15 g/l,at a final pH of 5.8. The spores were resuspended by adding 10 ml of a0.1% solution of Triton X-100 to each slant. The mycelium residues wereremoved by filtration through a nylon filter with pore size of 20 μm.The concentration of spores in the suspension was about 10⁶ spores/ml.

The method of mutation with EMS consisted of incubating 10⁶ spores/ml ina solution of EMS at 3% in 0.1 M sodium phosphate buffer pH 7.0 at roomtemperature for 60 minutes, achieving mortality rates of around 99%. Themutated spores were washed three times with 0.1% Triton X-100,centrifuging at 15° C. and 3000 rpm for 2 minutes.

The method of mutation with NTG consisted of incubating 10⁶ spores/ml ina solution that contained 250 82 g/ml of NTG and 0.1 M sodium citratebuffer pH 5.0 at room temperature for 30 minutes, achieving mortalityrates of around 95%. The mutated spores were washed three times with0.1% Triton X-100, centrifuging at 15° C. and 3000 rpm for 2 minutes.

The mutated spores were used for seeding Petri dishes that containedSutter IV solid medium supplemented with 0.1% Triton X-100. Thecomposition per liter of the Sutter IV medium is as follows: 40 gglucose, 4 g L-asparagine, 10 g KH₂PO₄, 40 ml of solution of traceelements 50x, and 30 g of agar. The solution of trace elements 50x ismade up of: 25 g/l of MgSO₄.7H₂O, 1.82 g/l of CaCl₂.2H₂O, 0.05 g/l ofthiamine, 0.1 g/l of citric acid, 0.075 g/l of Fe(NO₃)₃.9H₂O, 0.05 g/lof ZnSO₄.7H₂O, 0.17 g/l of MnSO₄.H₂O, 0.025 g/l of CuSO₄.5H₂O and 0.025g/l of NaMoO₄. 2H₂O. The seeded dishes were incubated at 25° C. for 4days to obtain isolated colonies.

EXAMPLE 2

Strategies for Selecting Mutants of B. trispora (−) that are LycopeneOverproducers

This example describes strategies for selecting strains of B. trispora(−) that are lycopene overproducers, based on (i) the use of trisporicacids and (ii) the color intensity of the colony. FIG. 1 shows thephylogeny of the B. trispora (−) strains used in the present invention.

Selection of lycopene-producing mutants by adding trisporic acids waseffected by placing sterile filters about 0.6 mm in diameter,impregnated with trisporic acids, on the colonies obtained from mutatedspores. The trisporic acids were obtained by extracting the supernatantfrom a mixed culture of the (+) and (−) strains of B. trispora with onevolume of chloroform after acidifying the sample to pH 2. The organicfraction was extracted with one volume of a 4% solution of sodiumbicarbonate, collecting the aqueous phase, which was acidified andextracted with chloroform again. Next, the chloroform was evaporated todryness and the residue, enriched with trisporic acids, was dissolved inethanol. The trisporic acids were quantified by measuring the absorbanceat 325 nm and assuming an absorption coefficient of 70 ml×mg⁻¹×cm⁻¹(Sutter R. P., Capage D. A., Harrison T. L., Keen W. A. 1973. J.Bacteriology 114:1074-1082).

The sterile filters were incubated in a solution of 1.2 mg/ml oftrisporic acids in ethanol and were then left to dry at room temperaturein sterile conditions. Next, the filters were placed on the mutantcolonies previously grown for 4 days at 25° C. The dishes were incubatedat 25° C. for a further 3 days, and it was observed that thelycopene-producing mutants became a deep red in color, in contrast tothe producers of β-carotene whose color was orange.

Applying this method with the CMA3 (−) strain, the mutant LMA1 (−) wasobtained (FIG. 1), which might have a mutation in the caRP gene, whichcodes for the enzyme lycopene cyclase and therefore, instead ofproducing β-carotene, should accumulate the intermediate lycopene duringthe process of fermentation of carotenoids. Therefore the LMA1 strain isable to produce lycopene without the need to add specific inhibitors oflycopene cyclase activity (example 5).

Selection of lycopene-producing mutants in relation to the colorintensity of the colony was effected as follows: The CMA1 strain(producer of β-carotene; see FIG. 1) was mutated as described inexample 1. The mutated spores were seeded on dishes of YEPDA solidmedium (bacto-peptone 20 g/l, yeast extract 10 g/l, glucose 20 g/l andagar 20 g/l, to a final pH of 6.0), and were incubated at 25° C. for 24hours and then at 20° C. for 48-72 hours. Finally, those colonies with adeeper yellow-orange color than the CMA1 (−) parent strain wereselected. In this way, 2 colonies were isolated with deep yellow-orangecolor (designated CMB1 (−) and CMB2 (−)). The CMB1 and CMB2 strainsmight be overproducers of lycopene in mixed fermentations with additionof specific inhibitors of lycopene cyclase activity (for exampleimidazole; example 4).

EXAMPLE 3

Strategies for Selecting Mutants of B. trispora (+) that areOverproducers of Lycopene

Selection of lycopene-overproducing mutants of B. trispora (+) waseffected using mutated spores in the manner described in example 1.These spores were seeded on Petri dishes that contained Sutter IV solidmedium supplemented with 0.1% imidazole and were incubated at 25° C. for7 days to obtain isolated colonies. Next, a portion from each of thecolonies was transferred to a dish of PDA on which B. trispora (−) hadbeen seeded previously. The distance between the seeding points of the(+) and (−) strains must be approximately 2 cm. The level of productionof lycopene in solid medium is estimated from the intensity ofcoloration in the zone of intersection of the colony of the (+) strainwith that of the (−) strain. In this way the B. trispora strain CPA1 (+)was selected, and this gave rise to a higher yield of lycopene in mixedsolid cultures with a series of (−) strains. The level of productionfrom the B. trispora strain CPA1 (+) was then analyzed in mixed culturein a liquid medium as described in examples 4 and 5. Scheme 3 shows thephylogeny of the B. trispora (+) strains used in the present invention.

EXAMPLE 4

Method of Production of Lycopene in A Flask by Mixed Culture of the (+)and (−) Strains of B. trispora by Adding Inhibitors of the EnzymeLycopene Cyclase

The (+) and (−) strains of B. trispora selected as described in examples1, 2 and 3 were fermented in a flask with the aim of determining thelevel of production of lycopene in a liquid medium and mixed culture.For this, an inoculum medium was prepared with the following compositionper liter: 23 g of soya flour, 47 g of maize flour, 0.5 g of KH₂PO₄,0.002 g of thiamine hydrochloride and pH adjusted to 6.3. The CPA1 (+)strain of B. trispora was seeded in 500-ml flasks containing 67 ml ofmedium at the rate of 103 spores per ml. The CMB2 (−) strain of B.trispora was seeded in 500-ml flasks containing 100 ml of medium at arate of 104 spores per ml. Both types of inoculum were incubated at 25°C. and 250 rpm for 44 hours.

On completion of incubation, the inocula of the (+) and (−) strains weremixed in the ratio 1/10 (v/v), and the mixture was used for inoculating250-ml flasks containing 20 ml of fermentation medium at a rate of 4 mlof the mixture of strains per flask. These flasks were incubated at 25°C. and 250 rpm for 5-6 days. The fermentation medium used had thefollowing composition per liter: 44 g of soya flour, 19 g of maizeflour, 5.5 g of KH₂PO₄, 0.002 g of thiamine hydrochloride, 100 ml ofvegetable oil, and pH adjusted to 7.5. The medium was distributed in250-ml flasks, which were inoculated with 20% of a mixture of the (+)and (−) strains of B. trispora. Between the 0th and the 36th hours offermentation, an inhibitor of the enzyme lycopene cyclase was added withthe aim of blocking. the biosynthetic pathway at the lycopene level (forexample, 0.75 mg/ml of imidazole). The flasks were incubated at 25° C.and 250 rpm for 6 days. At the end of fermentation, a mixture offermentation medium, glass beads and methylene chloride/methanol (1/1)was prepared. The mycelium of B. trispora was lyzed by vortex agitation,releasing the intracellular lycopene. The lycopene extracted with themethylene chloride/methanol mixture (ratio 1:1) was diluted in acetone.The concentration and purity of the lycopene were determined usingreversed-phase HPLC.

The yield obtained in mixed fermentations of the strains B. trisporaCPA1 (+) and B. trispora CMB2 (−) was 3 g/l of lycopene in the presenceof imidazole (FIG. 1).

EXAMPLE 5

Method of Production of Lycopene in the Flask by Mixed Culture of the B.trispora CPA1 (+) and B. trispora

LMA1 (−) strains without addition of inhibitors of the enzyme lycopenecyclase The strains of B. trispora LMA1 (−) and CPA1 (+) selected asdescribed in examples 1, 2 and 3 were fermented in a flask with the aimof determining the level of production of lycopene in liquid medium andmixed culture. For this, inocula were prepared from the (+) and (−)strains and fermentation was carried out in a flask as described inexample 4. The difference is that in this case the chemical inhibitor oflycopene cyclase activity was not added. At the end of fermentation,production of lycopene was evaluated as described in example 4.

The yields obtained by mixed fermentation of the strains B. trisporaCPA1 (+) and B. trispora LMA1 (−) were 1.2 g/l of lycopene in theabsence of imidazole (FIG. 2).

EXAMPLE 6

Method of Production of Lycopene in A Semi-Commercial Fermenter by MixedCulture of the (+) and (−) Strains of B. trispora with Addition ofInhibitors of the Enzyme Lycopene Cyclase

The CPA1 (+) and CMB2 (−) strains of B. trispora selected as describedin examples 2 and 3 were cultivated in a semi-commercial fermenter withthe aim of determining the lycopene yield. For this, an inoculum wasprepared with the following composition per liter: 23 g of soya flour,47 g of maize flour, 0.5 g of KH₂PO₄, 0.002 g of thiamine hydrochloride,and with its pH adjusted to 6.3. The (+) and (−) strains were seededseparately in 2000-ml flasks containing 500 ml of medium and wereincubated at 25° C. and 250 rpm for 44-48 hours.

Each of the strains was transferred to an intermediate growing tankcontaining a culture medium with the following composition per liter: 29g of Pharmamedia, 47 g of maize flour, 0.5 g of KH₂PO₄; 0.002 g ofthiamine hydrochloride and 1 g of antifoaming agent, and with its pHadjusted to 6.0. After incubating for 36-48 h, the (+) and (−) strainswere mixed in a 1/10 ratio and 20% of the mixture was used for seedingthe fermentation base medium, which had the following composition perliter: 44 g of soya flour, 19.25 g of maize flour, 0.55 g of KH₂PO₄,3.36 g of Na₂HPO₄, 0.184 g of NaH₂PO₄, 0.0022 g of thiaminehydrochloride, 100 g of vegetable oil and 0.175 g of antifoaming agent,and its initial pH was adjusted to 7.5. The fermentation was incubatedfor 100-140 hours at a temperature of 25-28° C. with stirring varyingbetween 150 and 250 rpm and aeration of 1-1.5 v/v/m. Between the 25thand 35th hours of fermentation, sterile imidazole was added to a finalconcentration of 0.75 g/l.

Evaluation of the concentration and purity of the lycopene at the end offermentation was carried out as described in example 4. The averagevalue of lycopene yield obtained in a series of different fermentationsof the CPA1 (+) and CMB2 (−) strains was 3.4 g/l (FIG. 2).

EXAMPLE 7

Method of Production of Lycopene in a Semi-Commercial Fermenter by MixedCulture of the Strains B. trispora

CPA1 (+) and B. trispora LMA1 (−) without addition of inhibitors of theenzyme lycopene cyclase The CPA1 (+) and LMA1 (−) strains of B. trisporaselected in the manner described in examples 2 and 3 were cultivated ina semi-commercial fermenter with the aim of determining the level ofproduction of lycopene without adding inhibitors of the enzyme lycopenecyclase. For this, an inoculation medium was prepared with the followingcomposition per liter: 23 g of soya flour, 47 g of maize flour, 0.5 g ofKH₂PO₄, 0.002 g of thiamine hydrochloride, and with its pH adjusted to6.3. The (+) and (−) strains were seeded separately in 2000-ml flaskscontaining 500 ml of medium and were incubated at 25° C. and 250 rpm for44-48 hours.

Each of the strains was transferred to an intermediate growing tankcontaining a culture medium with the following composition per liter: 29g of Pharmamedia, 47 g of maize flour, 0.5 g of KH₂PO₄; 0.002 g ofthiamine hydrochloride and 1 g of antifoaming agent, and with its pHadjusted to 6.0. After incubating for 36-48 h, the (+) and (−) strainswere mixed in a 1/10 ratio and 20% of the mixture was used for seedingthe fermentation base medium, which had the following composition perliter: 44 g of soya flour, 19.25 g of maize flour, 0.55 g of KH₂PO₄,3.36 g of Na₂HPO₄, 0.184 g of NaH₂PO₄, 0.0022 g of thiaminehydrochloride, 100 g of vegetable oil and 0.175 g of antifoaming agent,and its initial pH was adjusted to 7.5. The fermentation was incubatedfor 100-140 hours at a temperature of 25-28° C. with stirring varyingbetween 150 and 250 rpm and aeration of 1-1.5 v/v/m.

Evaluation of the concentration and purity of the lycopene at the end offermentation was carried out as described in example 4. The averagevalue of lycopene yield obtained without addition of imidazole in aseries of different fermentations of the CPA1 (+) and LMA1 (−) strainswas 1.6 g/l (FIG. 2).

EXAMPLE 8

Method of Recovering Lycopene by Resuspension of the Biomass in Alcohol

Three liters of fermentation medium were harvested, corresponding to abiosynthesis process in which the biosynthetic pathway was interruptedat the lycopene level. The titer of the medium was 3 g of lycopene perliter. The biomass of this culture medium was recovered by filtrationwith a Buchner funnel (porcelain filter funnel which supports a disk ofpaper or card which acts as a filtering sheet), obtaining 750 g of wetbiomass. The wet biomass was resuspended in 5.2 1 of azeotropicisopropanol 85/15 and was stirred for 30 minutes at 45±5° C. Recovery ofthe purified biomass using a Buchner funnel was repeated. This biomasswas dried under vacuum in a stove at a temperature below 45±5° C. for 18hours, until the content of residual solvents/water was less than 8%.150 g of dry, purified biomass was obtained with a lycopene contentequivalent to an assay value of 5.5%. The dry biomass was milled in aball mill and a 1 mm screen, obtaining a solid with the same percentagecontent, which was conditioned to permit solvent extraction.

Extraction was effected by mixing the 150 g of milled biomass with 2500ml of isobutyl acetate at 70±5° C., continuing stirring for 5 minutes.The spent biomass was separated from the lycopene-rich solvent byfiltering on a filter plate. The spent biomass was washed with 300 ml ofhot isobutyl acetate on the same filter, mixing the washing solvent withthe filtrate. All of the lycopene-rich isobutyl acetate was concentratedunder vacuum, keeping the temperature below 45±5° C., until the volumewas reduced to 300 ml, whereupon some of the lycopene crystallized. Inorder to complete crystallization and obtain a purer lycopene, 900 ml ofisopropanol was added. Stirring of the mixture was continued, undernitrogen and in the temperature range 0-5° C., for 3 hours. It wasfiltered in a Buchner funnel, washing the crystals with 25 ml ofisopropanol on the Buchner funnel. The crystals were collected and thendried under vacuum, obtaining 6.5 g of lycopene crystals with aspectrophotometric purity of 95%. Neither the presence of othercarotenoids, nor of cis lycopene, was detected by HPLC.

EXAMPLE 9

Method of Formulation of Lycopene in Oily Suspension

A laboratory ball mill, type Minizeta 003 from Netzsch, was charged withthe following, in this order: microspheres with diameter of 0.5-0.75 mm,23.5 g of sunflower oil (Koipe), 0.065 g of D,L-alpha-tocopherol (Merck)and the 6.5 g of crystalline lycopene obtained as described in example8. The mixture was milled at 3000 rpm for 5 minutes, obtaining 25 g of aviscous liquid of a deep reddish-purple color. Spectrophotometricanalysis of the oily suspension revealed a lycopene content of 21%.Neither the presence of other carotenoids nor of cis isomers of lycopenewas detected by HPLC. The crystals were smaller than 10 microns.

EXAMPLE 10

Method of Recovery of Lycopene by Direct Treatment of the FermentationMedium with Alcohol

1500 1 of lycopene fermentation medium (lycopene strength 2.3 g/l) wasmixed directly with 4500 liters of 85/15 isopropanol/water azeotrope.After stirring for 30 min at 45±5° C., the biomass was separated fromthe liquid using a centrifugal decanter. Around 250 kg of wet, purifiedbiomass was collected.

This biomass, soaked with water and isopropanol, was dried in a rotarydryer under vacuum until the content of residual solvents/water wasbelow 8%. The drying temperature was 45±5° C., and the average residencetime in the dryer was 14 hours. 85 kg of dry biomass was obtained with alycopene content equivalent to a specific concentration of 3.75%.

The dry biomass was extruded in a compactor, Hutt-Compacktor from BEPEX,obtaining a solid with the same specific concentration, which wasconditioned to permit solvent extraction.

Extraction was effected by mixing the 85 kg of milled solid with 1650 lof isobutyl acetate. The mixture was heated in line at 60±5° C. for anapproximate average contact time of 2 minutes and the spent biomass wasseparated from the lycopene-rich solvent using a centrifugal decanter.The whole of the lycopene-rich isobutyl acetate was concentrated undervacuum, maintaining the temperature below 45±5° C., until the volume wasreduced to 100 1, whereupon a proportion of the lycopene crystallized.To complete crystallization of the lycopene, 300 1 of isopropanol wasadded. The mixture was stirred for 3 h at 0-5° C. It was filtered on aBuchner funnel, collecting the lycopene crystals, which were dried undervacuum at room temperature. 2 kg of product was obtained, with aspectrometric purity of 96%. Neither the presence of other carotenoidsnor of cis isomers was detected by HPLC.

EXAMPLE 11

Method of Formulation of Water-Dispersible Lycopene using IsobutylAcetate as Solvent

3.5 g of lycopene obtained as described in example 10 was resuspended in410 ml of isobutyl acetate and 0.35 g of D,L-alpha-tocopherol (Merck)was added. The mixture was heated to boiling (114° C.) for 5 minutes,ensuring complete dissolution of the lycopene. At the same time, 12 g ofHi-Cap 100 (National Starch) and 12 g of Purity Gum 2000® (NationalStarch) were dissolved in 325 ml of demineralized water. The hot organicphase was emulsified for 5 minutes in one stage over the aqueous phaseusing an Ultraturrax emulsifier from IKA, achieving an average micellesize of 1.2 microns, measured with a Coulter LS230 analyzer. Theemulsion was transferred to a vacuum distillation system, adding 600 mlof water, so that the 410 ml of isobutyl acetate was evaporated withapproximately 700 ml of water. 203 g of liquid formulation (12.75% ofdry matter) was obtained, with a lycopene content of 1.25% (9.8% basedon the dry mass). Using HPLC, a content of cis lycopene of 23.3% wasdetected, but no other carotenoids were detected. This liquidformulation was atomized in a Büchi 190 laboratory atomizer, employing agas temperature of 190° C. at inlet and 90° C. at outlet, obtaining apowder of a deep red color, with a lycopene content of 8.4% and a watercontent of 6.5%. Using HPLC, a content of cis lycopene of 23% wasdetected, but no other carotenoids were detected.

EXAMPLE 12

Method of Formulation of Water-Dispersible Lycopene using IsobutylAcetate as Solvent

3.5 g of lycopene obtained as described in example 10 was resuspended in410 ml of isobutyl acetate and 0.35 g of D,L-alpha-tocopherol (Merck),0.7 g of ascorbyl palmitate (Merck) and 3.5 g of sunflower oil (Koipe)were added. The mixture was heated to boiling (114° C.) for 5 minutes,ensuring complete dissolution of the lycopene. At the same time, 10 g ofHi-Cap 100 (National Starch) and 10 g of Purity Gum 2000® (NationalStarch) were dissolved in 325 ml of demineralized water. The hot organicphase was emulsified for 5 minutes in one stage over the aqueous phaseusing an Ultraturrax emulsifier from IKA, achieving an average micellesize of 1.4 microns, measured with a Coulter LS230 analyzer. Theemulsion was transferred to a vacuum distillation system, adding 600 mlof water, so that the 410 ml of isobutyl acetate was evaporated withapproximately 700 ml of water. 195 g of liquid formulation (13.25% ofdry matter) was obtained, with a lycopene content of 1.3% (9.8% based onthe dry mass). Using HPLC, a content of cis lycopene of 25% wasdetected, but no other carotenoids were detected. This liquidformulation was atomized in a Büchi 190 laboratory atomizer, employing agas temperature of 190° C. at inlet and 90° C. at outlet, obtaining apowder of a deep red color, with a lycopene content of 8.5% and a watercontent of 6.0%. Using HPLC, a content of cis lycopene of 24.5% wasdetected, but no other carotenoids were detected.

EXAMPLE 13

Method of Formulation of Water-Dispersible Lycopene usingDichloromethane as Solvent

7.5 g of crystalline lycopene obtained as described in example 10 wasresuspended in 500 ml of dichloromethane, adding 0.75 g ofD,L-alpha-tocopherol (Merck), and heating the mixture at 35° C. for 5minutes. At the same time, 27 g of Hi-Cap 100 (National Starch) and 27 gof Purity Gum 2000® (National Starch) were dissolved in 400 ml ofdistilled water. The organic phase was emulsified for 15 minutes in onestage over the aqueous phase using an Ultraturrax emulsifier from IKA,achieving an average micelle size of 0.4 microns, measured with aCoulter LS230 analyzer. The emulsion was transferred to a vacuumdistillation system, adding 600 ml of water, so that the 500 ml ofdichloromethane was evaporated with approximately 600 ml of water. 400 gof liquid formulation (13.1% of dry matter) was obtained, with alycopene content of 1.5% (11.5% based on the dry mass). Using HPLC, acontent of cis lycopene of 6.5% was detected, but no other carotenoidswere detected. This liquid formulation was atomized in a Büchi 190laboratory atomizer, employing a gas temperature of 190° C. at inlet and90° C. at outlet, obtaining a powder of a deep red color, with alycopene content of 10.6% and a water content of 5.3%. Using HPLC, acontent of cis lycopene of 6.4% was detected, but no other carotenoidswere detected.

EXAMPLE 14

Method of Formulation of Water-Dispersible Lycopene usingDichloromethane as Solvent

7.5 g of crystalline lycopene obtained as described in example 10 wasresuspended in 500 ml of dichloromethane, adding 0.75 g ofD,L-alpha-tocopherol (Merck), and heating the mixture at 35° C. for 5minutes. At the same time, 27 g of Hi-Cap 100 (National Starch) and 27 gof Purity Gum 2000® (National Starch) were dissolved in 400 ml ofdistilled water. The organic phase was emulsified for 60 minutes in onestage over the aqueous phase using an Ultraturrax emulsifier from IKA,achieving an average micelle size of 0.23 microns, measured with aCoulter LS230 analyzer. The emulsion was transferred to a vacuumdistillation system, adding 600 ml of water, so that the 500 ml ofdichloromethane was evaporated with approximately 650 ml of water. 350 gof liquid formulation (14.4% of dry matter) was obtained, with alycopene content of 1.6% (11.4% based on the dry mass). Using HPLC, acontent of cis lycopene of 20% was detected, but no other carotenoidswere detected. This liquid formulation was freeze-dried in a laboratoryunit for 24 hours, obtaining a fluffy powder of a deep red color, with alycopene content of 10.7% and a water content of 7.4%. Using HPLC, acontent of cis lycopene of 15% was detected, but no other carotenoidswere detected.

DETAILED DESCRIPTION OF THE DIAGRAMS

FIG. 1. Production of lycopene by mixed fermentation of the B. trisporaCPA1 (+) strain in a flask with each of the following strains of B.trispora (−): L25, CMA1, CMA2, CMA3, CMA4, CMB1, CMB2 and LMA1 . Exceptin CPA1 (+)/LMA1 (−) mixed fermentation, imidazole was added asinhibitor of the enzyme lycopene cyclase. Ordinate: % of production withcontrol strain L25 (−) (VKPM F-744).

FIG. 2. Production of lycopene by mixed fermentation of the B. trisporaCPA1 (+) strain in a fermenter with each of the following strains of B.trispora (−): L25, CMA1, CMA2, CMA3, CMB1, CMB2 and LMA1. Except in CPA1(+)/LMA1 (−) mixed fermentation, imidazole was added as inhibitor of theenzyme lycopene cyclase. Ordinate: % of production with control strainL25 (−) (VKPM F-744).

1. Process of production of lycopene from biosynthetic sources,characterized in that it comprises the following steps in succession: a.Mixing (+) and (−) lycopene overproducer strains of Blakeslea trispora ,whose ages of vegetative stage of growth has been controlled, in aculture medium biomass in suitable fermentation conditions for producinglycopene; b. Treatment of the culture medium biomass of step a) withalcohol and separation of a wet, purified biomass; c. Conditioning ofthe wet, purified biomass by drying and disintegration or breaking; d.Solid-liquid extraction of the lycopene contained in the purifiedbiomass with an organic solvent, controlling the temperature and thetime of dissolution in function of the organic solvent used; e.Concentration of the enriched lycopene extract; fPrecipitation/crystallization of the lycopene from the concentratedextract by addition of alcohol; g. Filtration and drying, h.Formulation.
 2. Process according to claim 1, in which the strains usedas culture inoculum have an age ranging 30-60 h, preferably 48 h, with anumber of spores seeded of 800-1000 spores/ml for (+) strains and40000-60000 spores/ml for (−) strains, being seeded 0.1% v/v of eachinoculum in the primary culture phase.
 3. Process according to claim 2,in which the age of the primary cultures ranges 30-60 h, preferably36-48 h.
 4. Process according to claim 1, characterized in that in thestep a., together with the (+) strains, use is made of Blakesleatrispora (−) strains, mutants or transformants derived from them, whichproduce lycopene, preferably those described in scheme
 2. 5. Processaccording to claim 1, characterized in that in the step a., togetherwith the (−) strains, use is made of Blakeslea trispora (+) strains,mutants or transformants derived from them, which produce lycopene,preferably those described in scheme
 3. 6. Process according to claim 4in which the (−) and (+) strains of Blakeslea trispora mixed in theculture medium biomass are, respectively, VKPM F-744 and VKPM-F8 16 orlycopene overproducer strains selected thereof
 7. Process according toclaim 1, characterized in that in step a. mixed cultures of strains ofBlakeslea trispora (−) and (+) or mutants or transformants derived fromthem, which produce lycopene, are used in the absence of compounds thatare inhibitors of lycopene cyclase activity.
 8. Process according toclaim 1, characterized in that in step a. mixed cultures of strains ofBlakeslea trispora (−) and (+) or mutants or transformants derived fromthem, which produce lycopene, are used in the presence of compounds thatare inhibitors of lycopene cyclase activity.
 9. Process according toclaim 8, characterized by the addition of imidazole during thefermentation process, as an inhibitor of lycopene cyclase activity. 10.Process according to claim 9, characterized in that imidazole is addedbetween the 0th and 50th hours of fermentation, preferably between the25th and 35th hours.
 11. Process according to claim 9, characterized inthat the concentration of imidazole is maintained between 0.7 and 0.8g/l, preferably at 0.75 g/l.
 12. Process according to claim 8,characterized in that at least 3.4 g/1 of lycopene is produced at days5-6 of culture, in a semi-commercial or pilot fermenter.
 13. Processaccording to claim 6, characterized in that at least 1.6 g/l of lycopeneis produced at days 5-6 of culture, in a semi-commercial or pilotfermenter.
 14. Process according to claim 1, characterized in that: i)inocula of the (+) strain of B. trispora are seeded in a range of800-1000 spores/ml; ii) inocula of the (−) strain of B. trispora areseeded in a range of 10000-60000 spores/ml; iii) the inocula of the (+)and (−) strains of B. trispora are cultivated for at least 48 hours at25° C., iv) phases of primary culture of the (+) strain of B. trisporaare seeded with at least 0.1% (v/v) of the inoculation phase; v) phasesof primary culture of the (−) strain of B. trispora are seeded with atleast 0.1% (v/v) of the inoculation phase; vi) the primary phases of the(+) strain of B. trispora are cultivated for 36-48 hours at 26° C.; vii)the primary phases of the (−) strain of B. trispora are cultivated for36-48 hours at 28° C.; viii) the primary phases of the (+) and (−)strains of B. trispora are mixed in the proportions 1 (+)/10 (−) (v/v),ix) each fermenter is seeded with 10-20% (v/v) of the mixture of the (+)and (−) strains of B. trispora described in paragraph viii; x) the mixedculture is incubated for 5-6 days at 26° C.
 15. Process according toclaim 14, characterized in that at least 3.6 g/l of lycopene is producedat days 5-6 of culture.
 16. Process according to claim 15, characterizedin that at least 30 mg of lycopene is produced per gram of dry weight ofmycelium at days 5-6 of culture.
 17. Process according to claim 1,characterized in that step b. is carried out by previously separatingthe biomass from the culture medium and resuspending said biomass inalcohol, with an alcohol/biomass ratio of the order of 1 ml/g to 10ml/g.
 18. Process according to claim 1, characterized in that, in stepb., alcohol is used at a temperature between 0° C. and thatcorresponding to its boiling point, preferably between 10° and 50° C.19. Process according to claim 1, characterized in that step b. iscarried out directly by mixing the fermentation culture mediumcontaining the biomass without separating previously said biomass, inalcohol, with a culture medium/alcohol ratio between 1/0.5 and 1/5,preferably between 1/1 and 1/3, at a temperature between roomtemperature and the boiling point of the mixture, preferably betweenroom temperature and 60° C.
 20. Process according to claim 1,characterized in that a solvent of the ester type, preferably ethylacetate, propyl acetate, isopropyl acetate, n-butyl acetate or isobutylacetate, is used in step d. of solid-liquid extraction.
 21. Processaccording to claim 20, characterized in that a quantity of solvent ofthe order of 5 to 30 ml is used per g of the biomass resulting in stepc.
 22. Process according to claim 20, characterized in that when thesolvent used is isobutyl acetate the extraction is carried out attemperatures ranging 60-70±5° C.
 23. Process according to claim 22,characterized in that the solvent is used in such a way that the time ofcontact with the lycopene is less than 15 minutes, preferably 2-5minutes.
 24. Process according to claim 1, characterized in that, instep f., precipitation/crystallization of the concentrated product afterextraction is effected by adding a solvent in which the lycopene has lowsolubility, such as methanol, ethanol, propanol, or isopropanol, andwhich ensures that the substances of a lipophilic character accompanyingthe lycopene remain dissolved.
 25. Process according to claim 1, inwhich the formulation step h. consists of formation of amicrocrystalline suspension of lycopene by premixing the lycopenecrystals, antioxidants and oils in suitable proportions, followed bymilling.
 26. Process according to claim 25, in which the oil used is ofvegetable origin, preferably from sunflower, olive, maize, cottonseed orsoya.
 27. Process according to claim 25, characterized in that millingis carried out in a ball mill.
 28. Process according to claim 25,characterized in that liposoluble antioxidants are used, preferablynatural tocopherols, in a proportion from 0.2 to 15%, preferably 0.5-5%relative to the weight of the lycopene in the mixture.
 29. Processaccording to claim 1, in which formulation step h. comprises thepreparation of cold-water-dispersible (CWD) lycopene, by means ofProcess that encompasses the following steps: i) Dissolution of thecrystalline lycopene accompanied by antioxidant compounds in an organicsolvent; ii) Emulsion and/or microencapsulation of the aforesaid organicphase in an aqueous solution of modified starches; iii) Elimination ofthe solvent and some of the water by evaporation, obtaining the liquidformulation; iv) Drying and finishing.
 30. Process according to claim29, characterized in that methylene chloride is used as solvent in stepi. of dissolution of the lycopene.
 31. Process according to claim 29,characterized in that acyl esters are used as solvent in step i. ofdissolution of the lycopene, especially ethyl, propyl, isopropyl, nbutylor isobutyl acetates.
 32. Process according to claim 29, characterizedin that one or a mixture of antioxidants is used in step i. ofdissolution of the lycopene, preferably antioxidants of the tocopherolor ascorbyl palmitate type, in a proportion between 1-30%, preferably10-20%, relative to the weight of the lycopene.
 33. Process according toclaim 29, characterized in that in addition a vegetable oil is used instep i. of dissolution of the lycopene in the organic solvent. 34.Process according to claim 29, characterized in that modified starches,preferably in the form of esters, and more preferably octenyl succinateesters, are used as emulsifying and/or microencapsulating agents in stepii.
 35. Process according to claim 29, characterized in that step iv.the drying of the liquid formulation is effected by a process to beselected from: atomization by high-temperature pulverization,granulation by pulverization on a fluidized bed at relatively lowtemperature or freeze-drying.
 36. Process according to claim 35, inwhich the drying by granulation performed in step iv. is carried out byatomisation and the atomized product obtained thereof furtheragglomerated in a fluidised bed process by pulverization of a solutionof one of the modified starches or the suspension of microencapsulatedlycopene itself, thus obtaining a granulated product of increasedparticle size.
 37. Process according to claim 35, in which thegranulation comprises spraying a microencapsulated lycopene suspensionover a seed material, preferably inert material and more preferablyparticles of sugar or fine powder of previously granulated material. 38.Process according to claim 29, characterized in that, in step iv,finishing consists of coating the particles with aqueous solutions ofsugars or modified starches in a proportion of 0.5-10%.
 39. Crystallinelycopene obtainable by the process of claim 1, having a crystal purity,determined by spectrophotometric absorption of the crystals in n-hexaneat 472 nm (E1% 1cm=3450), is greater than 95%, the content of othercarotenoids being less than 3% and the content of cis lycopene beingalso less than 3%.
 40. Lycopene formulation consisting in an oilsuspension of the crystalline lycopene of claim 39, having a lycopenecrystal size below 10 microns, preferably less than 5 microns and morepreferably below 2 microns.
 41. Lycopene CWD formulation obtainable bythe process of claim 1 consisting in atomizatized lycopene microcrystalswith an average particle size between 10 and 100 microns.
 42. LycopeneCWD formulation obtainable by the process of claim 1 consisting in anagglomerated of atomized lycopene microcrystals with an average particlesize in the range 50-500 microns, preferably in the range 200-300microns.
 43. Lycopene CWD formulation obtainable by the process of claim1 consisting in granules of lycopene microcrystals with an averageparticle size between 100 and 2000 microns, preferably between 100 and800 microns and more preferably between 100 and 300 microns. 44.Lycopene CWD formulation according to claim 41 consisting in coatedparticles in which the coating was constituted by 0.5- 10% in dry weightof an aqueous solutions of sugars or modified starch.
 45. A methodcomprising using the crystalline lycopene of claim 39 as a colorant,especially in the food, pharmaceutical and cosmetics sectors.
 46. Amethod comprising using the crystalline lycopene of claim 39 as a dietsupplement.
 47. A method comprising using the formulation of claim 40 asa colorant, especially in the food.
 48. A method comprising using theformulation of claim 40 as a diet supplement.